1. Field of the Invention
The present invention provides heparin binding peptides for cardiovascular applications. More specifically, the present invention provides six related peptide sequences, all of which are designed to bind heparin and make a stable heparin/peptide complex, and antagonize the biological action(s) of heparin. The compounds of the present invention are useful as drugs given systemically (like protamine) or regionally or topically to antagonize or neutralize the anticoagulant activity of heparin. The compounds of the present invention are also useful in replacing protamine in insulin formulations for administration to diabetics.
2. Description of the Related Art
Heparin is a polydisperse, sulfated polysaccharide composed of alternating residues of N-glucoseamine and uranic acid (1). By nature of its synthesis, there is variability in the type of sugar backbone (iduronic vs. glucuronic acid), as well as in the degree and location of sulfated residues. Pharmaceutical grade heparin contains species which range in molecular weight from 6,000 to 20,000, and it is estimated that about 30% of the heparin by weight accounts for all its anticoagulant properties. Heparin, however, possesses numerous other biological properties, including the ability to inhibit smooth muscle cell proliferation (2), to catalyze lipoprotein lipase, to bind to endothelial cells, and to inhibit the interaction of von Willebrand factor (VWF) with platelets (3). Successful therapies based on these other activities have not yet been possible, mainly because the doses required to effect these other biological actions are associated with excessive anticoagulation. Nonetheless, it is well documented that heparin's ability to inhibit smooth muscle cell proliferation is distinct from its anticoagulant effects (2).
Heparin sulfate resembles heparin, but it is only poorly sulfated and has low anticoagulant activity. Dermatan sulfate, is also less sulfated than heparin and contains galactosamine in the saccharide backbone. Some of the residual anticoagulant properties of these latter two heparioids has been attributed to their catalysis of heparin cofactor H, rather than antithrombin III (6). However, the principal route of heparin anticoagulation is mediated through its interaction with antithrombin III (AT III).
Heparin binding to protein domains.
Complexation with heparin induces a conformational change in many proteins including ATIII (7-12), fibroblast growth factor (13,14), and mucous proteinase inhibitor (15). The guiding principle of heparin-protein interactions is that specific chemical unit structures within the heparin polymer bind tightly to structurally complementary specific domains within proteins (16-19). The present inventors have shown that the heparin binding domain of von Willebrand factor or AIII can be wholly replicated with synthetic peptides (16-21). Margalit et al (22) used a molecular modeling analysis of heparin binding domain sequences of proteins and peptides in the data base and showed that the spatial distribution of basic amino acids in all these heparin binding sequences conform to a motif wherein two basic residues (generally Arg) are separated by about 20 .ANG. facing opposite directions of an .alpha.-helix or .beta.-strand structure. Other cationic residues are interspersed between these two residues. Heparin may bind by wrapping itself around the peptide backbone, forming a coiled coil-like structure. Such a complex might easily induce a change in protein/peptide conformation. Fan et al., (23) and Tyler-Cross et al. (21) showed by mutational replacement and chemical synthesis strategies, respectively, that particular cationic residues within antithrombin III are essential for recognition and binding of heparin at the high affinity site; replacement or modification of these residues results in proteins (or peptides) which no longer bind heparin. The present inventors (21) suggested that ATIII Geneva, a naturally occurring mutant protein whose carriers display a predisposition toward thrombosis, results from a mutation of an essential Arg residue to Gin residue (24), which causes an unfavorable distortion in the conformation assumed by the heparin binding domain sequence.
The Need for a Heparin Antidote.
Heparin is used to render the blood incoagulable during open heart surgery, extracorporeal circulation, peripheral vascular surgery, percutaneous angioplasty and a multitude of other acute vascular interventions. Bleeding complications from heparin are especially common when the arterial tree is violated, occurring in as many as 10-15% of cases. Because of the toxicity and side effects of the only available antagonist, protamine, its use is primarily restricted to open heart surgery and emergencies. In most other acute, arterial applications of heparin, the anticoagulant effects are allowed to wane spontaneously over several hours. Many additional bleeding complications from heparin could be avoided if the anticoagulation caused by heparin could be more safely and tightly controlled. Thus, a heparin antidote is needed both to replace protamine and to use in more general applications where the toxicity of protamine has been prohibitive.
Protamine and its Problems.
The protamines, purified from fish (salmon) sperm, are a family of basic proteins rich in Arginine residues (25). Protamine neutralizes all of heparin's biologic effects by overwhelming the carbohydrate with cationic charges (26-28). The efficacy of protamine for heparin neutralization is thus related in part, to its total net cationic charge, but unfortunately, the toxicity of protamine is also related to its high charge density (29). Protamine administration in heparinized humans can frequently cause hypotension, pulmonary artery hypertension, myocardial depression, complement activation, thrombocytopenia, and leukopenia (30-36). Fatalities have been reported (37).
In cardiopulmonary bypass, protamine reversal of heparin is so essential that numerous clinical strategies have been devised to avoid side effects by administration in small or divided doses. This is a testament to the great clinical importance of this heparin antagonist. In spite of its poor therapeutic/toxic ratio, protamine has been used since 1939 (38) as the sole heparin antagonist available to clinicians.
Because endogenous and exogenous heparins can inhibit the proliferation of smooth muscle cells at sites of vascular injury (39-41), protamine is now implicated in another deleterious side effect. Edelman et al. (42) showed that protamine infusion negated the beneficial inhibitory effects of heparin on smooth muscle cell proliferation, and protamine alone exacerbated the proliferative response. These studies were performed in cell culture, and confirmed in whole animal studies. In each situation, they found that protamine negates the beneficial antiproliferative effects of heparin.
Thus, protamine may actually distort normal vascular repair by binding heparin or endogenous heparin-like molecules. These results argue strongly against the continued clinical use of protamine. In the setting of vascular injury or manipulation, such as arterial bypass or angioplasty, protamine administration may be especially harmful, leading to intimal hyperplasia, premature stenosis and thrombosis. A superior heparin antagonist is thus badly needed--one with more selective biologic actions and an improved safety profile.
There is growing commercial interest in safe protamine replacement drugs which would be used as heparin antagonists in elective or emergency procedures following cardiovascular surgery. In principal, this drug should specifically neutralize heparin's conventional anticoagulant properties without causing deleterious hemodynamic side-effects or exacerbation of the proliferative vascular response to injury.
The clinician's willingness to use Recombinant Platelet Factor 4 as a potential heparin antagonist is currently being assessed (43). Unfortunately, even though recombinant platelet factor 4 is effective in reversing heparin anticoagulation in the rat (44), in some non-rodent species its use caused severe adverse reactions, including anaphylaxis, and acute pulmonary vasoconstriction and hypertension, presumably associated with thromboxane release into the circulation (45). Moreover, platelet factor 4 has been identified as the definitive immunogen which complexes with heparin to cause heparin-induced thrombocytopenia (54-56) . This syndrome of immune sensitization to heparin (when complexed to platelet factor 4) is widely feared as it is associated with major morbidity and mortality. These new findings have arisen since the initial efforts to develop platelet factor 4 as a Protamine replacement, and raise serious questions as to its potential clinical use.
In yet another approach, Wakefield et al (46), continue to examine proteolytically derived fragments of protamine as potential Protamine replacement drugs. It is yet not clear whether relatively high molecular weight fragments derived from protamine will be less toxic than protamine itself, or whether such fragments can be produced on a commercial scale as potential pharmaceutics. Nor have they attempted to engineer any selectivity or specificity in their protamine substitutes.
A world-wide market clearly exists for a safe protamine replacement which would be a heparin antagonist for use following cardiovascular surgery, and in other applications.